Chronic Hyperinsulinism Induced Down-regulation of Insulin Post-Recentor Signaling Transduction in Hep G2 Cells

Summary: To study the regulatory effect of acute and chronic insulin treatment on insulin post-re-ceptor signaling transduction pathway in a human hepatoma cell line (Hep G2), Hep G2 cells wereincubated in the presence or absence of insulin with different concentrations in serum free mediafor 16 h and then stimulated with 100 nmol/L insulin for 1 min. Protein levels of insulin receptorβ-subunit (IRβ), insulin receptor substrate-1 (IRS-1) and p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) were determined in total cell lysates by Western-immunoblot. Phosphorylat-ed proteins IRβ, IRS-1 and interaction of PI 3-kinase with IRS-1 were determined by immunopre-cipitation. Results showed that 1-min insulin stimulation rapidly induced tyrosine phosphorylationof IRβ and IRS-l, which in turn, resulting in association of PI 3-kinase with IRS-1. 1-100 nmol/L chronic insulin treatment induced a dose-dependent decrease in the protein level of IRβ and aslight decrease in the protein level of IRS-1. There wass more marked reduction in the phospho-rylation of IRβ, IRS-1, reaching a nadir of 22 % (P＜0. 01) and 15 % (P＜0. 01) of control lev-els, respectively, after 16 h treatment with 100 nmol/L insulin. The association between IRS-1and PI 3-kinase was decreased by 66 % (P＜0. 01). There was no significant change in PI 3-ki-nase protein levels. These data suggest that chronic insulin treatment can induce alterations ofIRβ, IRS-1 and PI 3-kinase three early steps in insulin action, which contributes significantly toinsulin resistance, and may account for desensitization of insulin action.     

交泰丸通过骨骼肌磷脂酰肌醇(-3)激酶通路提高糖尿病大鼠胰岛素信号强度

Objective: To investigate the effect of Jiaotai Pill (交泰丸, JTP) at different constitutional proportions on insulin signaling through phosphatidylinositol 3-kinase (PI3K) pathway in the skeletal muscle of diabetic rats. Methods: The rat model of type 2 diabetes mellitus (T2DM) was established by intravenous injection of a small dose of streptozotoein plus high fat diet feeding. JTP at the same dosage of cinnamon and the increasing dosage of Coptis chinensis was administered to diabetic rats for nine weeks respectively. Plasma glucose and insulin levels were assayed. The expressions of proteins were determined by Western blot method. Results: All the three formulations of JTP decreased plasma glucose and fasting insulin levels as well as increased the protein expressions of insulin receptor β (InsRβ) subunit, insulin receptor substrate-1 (IRS-1), PI3K p85 subunit and glucose transporter 4 (GLUT4) in skeletal muscle. Meanwhile, JTP increased the tyrosine phosphorylation of InsRβ subunit and IRS-1, and reduced the serine phosphorylation of IRS-1 in skeletal muscle. Interestingly, the effect of JTP on improving insulin sensitivity was not dose-dependent. In contrast, JTP containing the least amount of Coptis chinensis exhibited the best effect. Conclusion: JTP at different constitutional proportions attenuates the development of diabetes in a rat model of T2DM. The mechanism might be associated with enhancing insulin signaling through PI3K pathway in the skeletal muscle.     

Influence of berberine on protein tyrosine kinase of erythrocyte insulin receptors from type 2 diabetes mellitus

Objective： Bererine has been used to treat type 2 diabetes mellitus in Chinese traditional medicine because of its hypoglycemic effect. In this report, we compared the intrinsic tyrosine kinase activities of erythrocyte insulin receptors from type 2 diabetes mellitus with or without stimulation by berberine in vitro. Methods- Preparations containing insulin receptors were obtained from soluble human erythrocytes, and the insulin receptors were partially purified by affinity chromatography. The tyrosine kinase activity was measured by the exogenous substrate phosphorylation. Results： Both the membrane tyrosine kinase activity and the purified receptor tyrosine kinase activity from diabetics decreased significantly compared with those of normal individuals （reduced by 67.4% and 47.2%, respectively）. After incubation with berbefine, there is a statistical difference in the activity of membrane tyrosine kinase for diabetic patients （ a 150% increase）. Berefine had no effect on the tyrosine kinase activity of purified insulin receptors. Conclusion： We concluded from these results that berbefine was able to improve the insulin sensitivity by increasing the protein tyrosine kinase activity of membrane-bound insulin receptors from type 2 diabetes mellitus.     

Effect of Metformin on Tyrosine Kinase Autophosphorylation of Erythrocyte Insulin Receptor from Women with Polycystic Ovary Syndrome

Objective To document the possible effect of tyrosine kinase autophosphorylation on erythrocyte insulin-receptor after 12 weeks of metformin administration from the women with polycystic ovary syndrome (PCOS). Methods Thirty non-obese (BMI<25 kg/m 2 ) healthy women with normal reproductive cycles were evaluated by conventional criteria as the control, 30 non-obese women with PCOS were categorized as nob-PCOS group, 40 obese women with PCOS (BMI ≥ 25 kg/m 2 ) were categorized as ob-PCOS group. Subjects with PCOS were given metformin 850 mg/d for 12 weeks. The autophosphorylation of insulin receptor pY1158, pY1162/1163 and insulin receptor-β subunit (IR-β) from the solubilised erythrocyte were detected by ELISA. Results 1) Only the autophosphorylation level of pY1158 in nob-PCOS and ob-PCOS groups was lower (P<0.05) than that in control group and it can be increased after insulin stimulation (P<0.05); no similar changes were found in pY1162/1163 (P>0.05), even insulin-stimulated insulin receptor (P>0.05) from both of PCOS groups. 2) After metformin administration, the pY1158, pY1162/pY1163 autophosphorylation levels were increased (P<0.01) and further increased after plus insulin stimulation in vitro (P<0.01). The pY1162/1163 autophosphorylation level was increased in nob-PCOS group (P<0.01) and ob-PCOS group (P<0.05). 3) No difference was found in concentration of IR-β between control group and both of PCOS groups following metformin administration (P>0.05). In summary, oral administration of metformin ledto an significant increase in tyrosine kinase activity in both groups of PCOS. Conclusion The impairment of tyrosine autophosphorylation at pY1158 may contribute to insulin resistance, the tyrosine kinase activity per receptor of solubilised erythrocytes were significantly increased by metformin administration and the effect of metformin on insulin-receptor tyrosine kinase activity appeared to be independent of either of these variables.     

Mechanism by which statins influence insulin signaling pathway

Recent studies have shown that statins can influence insulin resistance （IR） in animal models and inhumans.1.2 However, the mechanism by which statins influence the insulin signaling pathway （ISP） remains obscure. It has been proposed that the pleiotropic effects of statins might be involved in regulation of IR.The phosphatidylinositol 3-kinase （PI3K）-Akt/protein kinase B （PKB） pathway is the main ISE Insulin binding to insulin receptor initiates PI3K-Akt pathway by phosphorylates tyrosine residue of IR substrate proteins （IRSs） including IRS-1 and IRS-2. Activation of the PI3K leads to the accumulation of phosphatidylinositol 3,4,5-triphosphate （PIP3）. PIP3 activates serine/ threonine kinase （Akt）. Glucose transporter-4 （GLUT-4） translocates to plasma membrane from cytosol by serine phosphorylation of Akt and directly regulates glucose metabolism in liver, muscle and adipose tissue （Figure 1）. However, an overall mechanism by which statins influence ISP remained obscure.     

Vaccination with a Recombinant Chicken FGFR-1 Bypasses Immunological Tolerance against Self-FGFR-1 in Mice

It has been generally believed that that the growth and metastasis of solid tumors depend on tumor angiogenesis, the generation of new blood vessels. At present, basic fibroblast growth factor (bFGF) has been shown to be one of the most important angiogenic growth factors for tumor angiogenesis. bFGF serves its biological functions through interaction with its high-affinity receptor, fibro- blast growth factor receptor-1(FGFR-1)[1–3]. FGFR-1 is a member of the receptor tyrosine kinase co…     

A simple and efficient method of DNA extraction from formalin-fixed and paraffin-embedded tissues for screening EGFR gene mutation by gene sequencing

Epidermal growth factor receptor （EGFR） is frequently overexpressed in non-small-cell lung cancer （NSCLC） and plays a key role in tumorigenesis.1 Small molecule tyrosine kinase inhibitors （TKIs）,such as gefitinib,erlotinib,and icotinib,which can inhibit receptor tyrosine kinase activity of EGFR become clinically available for the treatment of non-small-cell lung cancer （NSCLC）.2 NSCLC patients with EGFR mutation have experienced a marked response to EGFR-TKIs therapy.3 Detection of mutations of the EGFR gene is critical for predicting the response to therapy with TKIs.4     

Genistein reverses free fatty acid-induced insulin resistance in HepG2 hepatocytes through targeting JNK

This study investigated the effects and molecular mechanisms of genistein in improving insulin resistance induced by free fatty acids (FFAs) in HepG2 hepatocytes. A model of insulin resistance in HepG2 cells was established by adding palmitic acid (0.5 mmol/L) to the culture medium and the cells were treated by genistein. Glucose consumption of HepG2 cells was determined by glucose oxidase method. The levels of c-jun N-terminal kinase (JNK) phosphorylation, insulin receptor substrate-1 (IRS-1) Ser307 phosphorylation, JNK, IRS-1, phosphatidylinositol-3-kinase p85 (PI-3K p85) and glucose transporter 1 (GLUT1) proteins were detected by Western blotting. The results showed that after the treatment with palmitic acid for 24 h, the insulin-stimulated glucose transport in HepG2 cells was inhibited, and the glucose consumption was substantially reduced. Meanwhile, the expressions of IRS-1, PI-3K p85 protein and GLUT1 were obviously reduced, while the levels of JNK phosphorylation and IRS-1 Ser307 phosphorylation and the expression of JNK protein were significantly increased, as compared with cells of normal control. However, the aforementioned indices, which indicated the existence of insulin resistance, were reversed by genistein at 1-4 μmol/L in a dose-dependent manner. It was concluded that insulin resistance induced by FFAs in HepG2 hepatocytes could be improved by genistein. Genistein might reverse FFAs-induced insulin resistance in HepG2 cells by targeting JNK.     

Upper respiratory tract,lung

208097 Construction of siRNA expression vector targeting IGF-1R and its ability to induce cell apoptosis in human lung cancer cells/Dong Aiqiang(董爱强,Dept Cardiothorac Surg,2nd Affil Hosp,School Med,Zhejiang Univ,Hangzhou 310009)…∥Chin J Thorac Cardiovasc Surg.-2007,23(4).-263～265Objective Insulin-like growth factor-1 receptor(IGF-1R),similar to insulin receptor ,is one of the families of receptor tyrosine kinases.,which has been found to be overexpressed in a variety of cancer.     

Role of protein tyrosine kinase in IL-1β induced activation of mitogen-activated protein kinase in fibroblast-like synoviocytes of rheumatoid arthritis

Objectives To study mitogen-activated protein kinase (MAPKs) activation in fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA) under the stimulation of IL-1β, and to elucidate the role of protein tyrosine kinase (PTK) in the activation of MAPKs. Methods Primary cultures of RA FLS were used. Western blot was applied to examine transient changes in protein tyrosine phosphorylation status and MAPKs activation in RA FLS stimulated with IL-1β at various doses, and over different periods. Genistein, the specific PTK inhibitor, was used to evaluate the inhibitory role in activation of MAPKs by IL-1β.Results IL-1β transiently increased protein tyrosine phosphorylation, and activated the MAPKs cascades (mainly ERK(2), JNK(2) and P(38)) in RA FLS. There was no obvious difference in MAPKs activation among different doses of IL-1β (1 IU/ml,10 IU/ml, 100 IU/ml), but the peak activation of ERK(2), JNK(2) and P(38)took place at 5 min, 15 min and 1 min, respectively, after stimulation with IL-1β. The activation of ERK(2)was inhibited by genistein, but the inhibitory role on that of JNK and P(38)was relatively weak. Conclusions During signal transduction of IL-1β in RA FLS, tyrosine phosphorylation was increased transiently, the MAPKs cascade was activated in a few minutes, and there was heterogenicity in the activation among three subfamily members. PTK had a role in the activation of ERK, but had weak effects on that of JNK and P(38).     

Leptin Regulated Insulin Secretion via Stimulating IRS2-associated Phosphoinositide 3-kinase Activity in the isolated Rat Pancreatic Islets

Leptin,theproductofob gene,isa peptidehor-mone secreted by adipocytes thatacts in the hypotha-lamus and playsa centralrole in regulation of feedingbehavior and energy homeostasis[1] .It has recentlybeen confirmed that leptin receptor(OB- R) m RNAand protein were expressed in rat pancreaticβ- cells,indicating thatleptin may directly regulate insulin se-cretion.Several groups have addressed this question,but the results were controversial[2 ,3] .Up to now,the molecular mechanism by which lept…     

氯沙坦改善3T3-L1脂肪细胞胰岛素抵抗的机制研究

目的探讨氯沙坦改善3T3-L1脂肪细胞胰岛素抵抗的主要作用机制。方法以地塞米松诱导3T3-L1脂肪细胞,建立胰岛素抵抗细胞模型,根据细胞模型添加干预药物的不同分为模型对照组（不添加任何药物）、氯沙坦组（分别给予1、10、100μmol/L氯沙坦干预48 h）和wortmannin＋氯沙坦组,wortmannin＋氯沙坦组以100 nmol/L的磷脂酰肌醇3激酶（PI3K）特异性抑制剂wortmannin预处理20 min后再加入100μmol/L氯沙坦干预48 h。观察脂肪细胞体积的变化,采用葡萄糖氧化酶法检测细胞培养上清液中葡萄糖的浓度,采用Western blotting分析脂肪细胞中PI3K和胰岛素受体底物1（IRS-1）的表达以及IRS-1丝氨酸磷酸化水平。结果与模型对照组比较,氯沙坦组脂肪细胞体积明显缩小（P〈0.01）,细胞培养上清液中葡萄糖的浓度显著降低（P〈0.01）,PI3K和IRS-1表达明显上升（P〈0.01）,IRS-1丝氨酸磷酸化水平显著下降（P〈0.01）但可被wortmannin阻断。结论氯沙坦可使3T3-L1脂肪细胞胰岛素抵抗模型的细胞体积缩小,并增加细胞对葡萄糖的利用,其机制可能与PI3K信号通路有关。     

Regulation of leptin on insulin secretion and sulfonulurea receptor 1 transcription level in isolated rats pancreatic islets

Objective To investigate the regulation of leptin on insulin secretion and expression of ATP-sensitive potassium channel subunit sulfonulurea receptor 1 (SUR1) mRNA, and to determine whether the effects of leptin are mediated through known intracellular signaling transduction.Methods Pancreatic islets were isolated by the collagenase method from male SD rats. The purified islets were incubated with different concentrations of leptin for 2 h in the presence of different concentrations of glucose. Insulin release was measured using radioimmunoassay. Expression of SUR1 mRNA was detected by RT-PCR.Results In the presence of leptin 2 nmol/L, insulin release was significantly inhibited at either 11.1 or 16.7 mmol/L glucose concentration (both P<0.05), but insulin release was not altered at glucose of 5. 6 mmol/L physiological concentration. The dose-response experiment showed that the maximal effect of leptin on insulin secretion achieved at 2 nmol/L. Exposure of islets to 2 nmol/L leptin induced a significan     

visfatin对体外脂肪细胞胰岛素受体底物和PI3K表达的影响

目的：从胰岛素经典信号传导通路磷脂酰肌醇-3-激酶（PI3K）角度探讨内脏脂肪素（visfatin）与2型糖尿病（T2DM）胰岛素抵抗（IR）的关系。方法：复苏、传代和诱导分化人源T2DM前脂肪细胞,构建 visfatin过表达载体,进行载体转化、培养和提取;以4个不同表达梯度（0.0、1.0、2.5和5.0 μg）转染传代脂肪细胞,以0.0 μg组为对照组,其余3组为观察组;Q-PCR法检测visfatin 、胰岛素受体底物1（IRS-1）、胰岛素受体底物2（IRS-2）和 PI3K（P85α） mRNA表达水平,Western blotting法检测visfatin、IRS-1、IRS-2和PI3K（P85α） 蛋白表达水平及IRS-1和IRS-2酪氨酸磷酸化水平,[³H]-2-脱氧-D-葡萄糖摄取法测定细胞葡萄糖摄取率的变化。结果：各组 visfatin mRNA及蛋白表达水平随转染浓度梯度升高而升高（P<0.01）,所构建visfatin过表达载体有效。随visfatin表达增加,各组IRS-1和PI3K（P85α） mRNA和蛋白表达水平以及IRS-1磷酸化程度均明显升高（P< 0.01）,但IRS-2 mRNA和蛋白表达水平未出现明显变化（P>0.05）。脂肪细胞的葡萄糖摄取率随visfatin表达增加而升高（P<0.05）。结论：体外脂肪细胞visfatin过表达可增加IRS-1和PI3K的表达水平。     

Potent Therapeutic Effects of Shouwu Jiangqi Decoction(首乌僵芪汤)on Polycystic Ovary Syndrome with Insulin Resistance in Rats

Objective:To investigate the effect of Shouwu Jiangqi Decoction(首乌僵芪汤,SJD) on polycystic ovary syndrome(PCOS) with insulin resistance(IR) in rats and to explore the underlining molecular mechanisms.Methods:A total of 51 female Sprague-Dawley rats were randomly divided into 6 groups:control group(n=7),model group(n=8),SJD high-dose group(n=9),SJD medium-dose group(n=9),SJD low-dose group(n=9) and DMBG group(n=9).Radioimmunoassay was used to measure serum follicle-stimulating hormone(FSH),luteinizing hormone(LH) and testosterone concentrations and qRT-PCR and western blot were used to examine the expression levels of mRNA and protein respectively of insulin receptor substrate 1(IRS-1)and phosphatidylinositide 3-kinases(PI3K) p85α in different groups.Results:FSH level significantly decreased in the model group compared with the normal control(P0.01),and high-dose SJD and DMBG can significantly increase FSH level(P0.01).LH level showed a mild increase without statistic significance in the model group compared with the control and different dosages of SJD had no significance effect on LH level,while DMBG can significantly decrease LH level(P0.01).Testosterone level significantly increased in the model group compared with the control group(P0.01),and high-dose SJD and DMBG can significantly decrease testosterone level(P0.01).The expression of IRS-1 as well as PI3Kp85α were significantly decreased in the model group compared with the normal control group at both mRNA(P0.001) and protein(P0.01) level,and both high-dose SJD and DMBG can enhance IRS-1 and PI3 K expression(P0.05).Conclusions:SJD has potent therapeutic effects on PCOS with IR in rats.The therapeutic effects of SJD on IR and ovulatory dysfunction are probably achieved through correcting the defective insulin signaling transduction.     

游离脂肪酸引起肝细胞胰岛素抵抗及其机制的研究

目的研究游离脂肪酸（FFA）诱导人肝癌细胞（HepG2）引起胰岛素抵抗及其分子机制.方法应用含0.25 mmol/L的软脂酸（PA组）或100 nmol/L胰岛素（Ins组）与不含软脂酸和胰岛素（正常组）的DMEM培养基培养HepG2细胞24 h,正常组和PA组中又分加与不加磷酯酰肌醇3激酶（PI3K）抑制剂Wort-mannin两个亚组,100 nmol/L胰岛素刺激后分别测定培养液中的葡萄糖浓度、细胞内的糖原含量、磷酸烯醇式丙酮酸羧激酶（PEPCK）活性及胰岛素受体底物2（IRS-2）的蛋白水平.结果PA组和Ins组培养液中葡萄糖含量显著高于正常组（P＜0.01）,而细胞内糖原含量显著减少（P＜0.01）;PA组胰岛素刺激的PEPCK活性显著高于正常组（P＜0.01）,IRS-2蛋白水平显著低于正常组（P＜0.01）.无论应用Wortmannin处理与否,PA组中的PEPCK活性及IRS-2蛋白水平差异无显著性（P＞0.05）,而正常组中PEPCK活性及IRS-2蛋白水平的差异有显著性（P＜0.01）.结论加0.25 mmol/L的软脂酸培养24 h后,肝细胞可能由于胰岛素信号转导障碍产生胰岛素抵抗;胰岛素抵抗的形成可能与IRS-2及PI3K相关分子缺陷有关.